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Bulgarian Journal of Agricultural Science, 14 (No 5) 2008, 449-453 Agricultural Academy

TRANSSEXUAL FORMS OF ( TEREBINTHUS L.) FROM BULGARIA – BIOTECHNOLOGICAL APPROACHES FOR PRESERVATION, MULTIPLICATION AND INCLUSION IN SELECTION PROGRAMS

P. GERCHEVA1, A. ZHIVONDOV1, L. NACHEVA1 and D. AVANZATO2 1 Fruit Growing Institute, BG – 4000 Plovdiv, Bulgaria 2 CRA-Instituto Sperimentale per la Frutticoltura, Roma, Italy Abstract

GERCHEVA, P., A. ZHIVONDOV, L. NACHEVA and D. AVANZATO, 2008. Transsexual forms of pista- chio (Pistacia terebinthus L.) from Bulgaria – biotechnological approaches for preservation, multiplication and inclusion in selection programs. Bulg. J. Agric. Sci., 14: 449-453

The present article includes a detailed survey of the application of the biotechnological approaches to the multiplication and study of the Pistacia (pistachio) – nut fruit crop not widespread in Bulgaria, which economic significance in Mediterranean region and around the world grows continuously. The main reason for the develop- ment of the culture is the high nourishing value of the fruits, the existence of wild species and the potential for inter- and interspecies hybridization and mainly the exceptional drought resistance of the kind which allows its cultivation in soils that are under no irrigation, small-productive, stony and sliding (pliant to erosion) soils. The Pistacia genus () includes 12 deciduous species, all of which are trees. These species are dioecious, anemophylic and have their male and female flowers on separate trees. Among them, only P. vera is domesticated. For the industrial plantations of pistachio there should be a male tree for every 8-11 female trees, thus 10 % from the pistachio plantation is non-productive. In the summer of 2002, Dr. D. Avanzato found a rare transsexual form of P. terebinthus in the Rhodopi Mountain. Later existence of a whole isolated population of these trees is determined. The discovered trees are the first case of a defined transsexual form among P. terebinthus. The study of the area where this transsexual form of Pistacia terebinthus was found and the common application of in vivo and in vitro methods for propagation and molecular markers will allow its preservation and use as a rootstock and as a donor for monoeciousness in the pistachio hybridization programs.

Key words: pistachio, monoecious forms, in vitro, micro propagation Abbreviations: BAP - 6-benzylaminopurine; IBA - indole-3-butyric acid; NAA - a-naphthaleneacetic acid; IAA - indole-3-acetic acid The economic importance of Pistacia (pistachio) production of nuts from pistachio has increased 5.4 in the Mediterranean region and around the world is times, only in that increase is 8.7 times, in the continuously growing. For the last 20 years the world USA – 7.8 times. The main reason for that develop- 450 P. Gercheva, A. Zhivondov, L. Nacheva and D. Avanzato ment is the high nourishing value of the fruits, the ex- ation of cultivars where the male and female racemes istence of wild species, the potential for in inter- and are in one tree and it has a high crop of quality fruits interspecies hybridization and mainly the astonishing would make unnecessary the need male trees to be drought resistance of the species that allows its culti- grown in the plantations and thus it could increase the vation in soils that are under no irrigation, small-pro- crop with about 10 %. To achieve that goal it is nec- ductive, stony and sliding (pliant to erosion) soils essary to find a donor of genes for monoeciousness in (Kaska, 2002). the pistachio. Until recently there were only three re- The pistachio grows mainly in regions between 30 ports of such trees in the literature. and 45 degree of latitude in the northern and southern Ozbek and Ayfer (1958) have found two hermaph- hemisphere and some other microclimatic zones in the rodites, probably hybrid trees of P. vera or hybrids different continents where there are suitable ecologic of P. vera and P. terebinthus in , where the conditions. It is from the Pistacia genus male and female organs are in one and the same blos- (Anacardiaceae) that includes more than 12 types of som. deciduous trees (Zohary, 1952). All of them are dio- The second report is that of Crane (1974), where ecious, anemophylic and have their male and female he describes three trees, hybrids between P. vera and flowers on separate trees. Among them only P. vera P. atlantica, where the male and female flowers are is domesticated, while P. integerrima, P. atlantica, on separate shoots. P. terebinthus, etc. have economic application as Kafkas et al. (2000) have reported of found wild rootstocks for the cultivars from P. vera. population of P. atlantica that consisted of several Pistacia terebinthus has significant importance monoecious trees in Yunt Mountains in Turkey. The and is widely spread in the whole Mediterranean re- authors used the found forms in experiments of inter- gion – Italy, Greece, Turkey, Tunisia, Syria, etc., as a and interspecies hybridization for creation of monoe- wild species and a rootstock in the plantations. The cious cultivars of pistachio and for clarification of the same species is a tree with thick crown and height up mechanism for determination of the sex in the species to 8 m. It has long, reddish and smooth shoots which from Pistacia genus (Kafkas, 2002). change to an ash colour at maturity. The grow In the summer of 2002 during a visit to the well in all types of soil, including rocky areas because Rhodopes Dr. D. Avanzato found a unique transsexual of their drought resistance. P. terebinthus crosses form of P. terebinthus (Avanzato, 2003; Avanzato easily and spontaneously with P. vera and the hy- and Quarta, 2004), and in subsequent common ex- brids have economic significance as pollen donors for peditions with Dr. Argir Zhivondov the existence of pistachio crops (Avanzato and Quatra, 2004). The an isolated population of such trees was discovered. female racemes of P. terebinthus are relatively short, The transsexual forms have the classical habitus of P. thick clusters with huge number of small flowers. The terebinthus. The first found tree is about 50 years male racemes (rachises) during the florescence achieve old, and the rest ones are significantly younger. The up to 80 mm. The florescence begins from the middle only determined difference is in the structure of the of the rachis and goes to the top and the base. It takes racemes (Buffa et al., 2007). The plants have both place in the second half of April. separate male and female racemes, situated on one- As we already mentioned, all plants from the year , and male and female racemes in one and Pistacia genus are dioecious, i.e. the male and female the same raceme. The found plants are first case of racemes are formed in different trees (Zohary, 1952). defined transsexuality of P. terebinthus. This fact has For industrial plantations of pistachio it is necessary a unusual scientific and practical importance. male tree to be plant for every 8-11 female ones From theoretic point of view it is a rare case of (Maranto and Crane, 1982), hence 10 % of the pis- defined monoeciousness among a typically dioecious tachio plantation is not productive. The eventual cre- species. From the fact that such form is found in a Transsexual Forms of Pistachio (Pistacia terebinthus l.) from Bulgaria..... 451 small isolated population of P. terebinthus can be In in vitro propagation of wild species from genus assumed that monoeciousness can be inherited in the Pistacia Behboodi (2002) examines the influence of generation of dioecious female plants. The compari- different growth regulators. The best results are son of monoecious and dioecious plants and their hy- achieved in the use of BAP and NAA. The results for brids by molecular markers could contribute to the P. vera cv. Mateur are analogous – BAP (2.0 mg/l) clarification of the mechanism for inheritance of that stimulates adventitious regeneration of shoots (Chatibi feature. et al., 1998a). Ghoraishi (2006) also mentions that From other point of view the discovery of these BAP in concentration 20 µM is the most efficient in unique forms has an important economic significance, the micropropagation of P. mutica. The author men- too. These forms give an opportunity for obtaining tions that treating with 100 µM IBA for 5 days signifi- interspecies hybrids with cultivars of P. vera and trans- cantly improves the rooting of the microplants. fer of that feature. Special attention is drawn to the optimization of The achievement of such ambitious task is pos- the salt content of the nutrient media for getting over sible only if there is a successful elaboration and ap- the browning of the shoot tip, the existence of red plication of biotechnological and molecular-genetic pigmentation on the leaves and improvement of the methods in the multiplication and selection of the spe- vitality of the plants (Chatibi et al., 1998b). cies. Ultrastructure analysis is used for characterization The use of tissue cultures for multiplication of the of the shoot-tip necrosis at in vitro cultivation of P. species from Pistacia genus becomes increasingly sig- vera (Abousalini and Mantell, 1995). Damages of the nificant in the last 20 years (Martinelli and Loretti, plasmalemma, desintegration of the membranes and 1988; Bargchi et al., 1989; Gonzales and Frutos, organelas and cell autolysis are observed. 1990; Mehlenbacher, 2003). In order the propagation to be accelerated in vitro There are reports of successful experiments of techniques based on the somatic embryogenesis are micropropagation of P. terebinthus and P. vera elaborated (Onay, 2000). Development of somatic where the oxidation of the media is controlled by ad- embryos from leaf explants (Onay, 2000), immature dition of ascorbic acid, and the most successful intro- embryos and female flowers (Onay et al., 2004) of P. duction of culture is achieved at one-month-old seed- vera is achieved. Regeneration from embryoids and lings (Gannoun et al., 1995). embryogenic masses (Onay et al., 1996) is reported. The main limiting factor in the introduction and sta- The influence of the species and the concentration of bilization of in vitro culture for many trees, as well as growth regulators, included in the nutrient media over the pistachio, is the browning of the explants as a re- the formation and growth of the somatic embryos and sult of the increased content of phenols. Tabiyeh et al. the germination of the plants is examined (Onay et al., (2006) report of decrease in the oxidation and im- 2004). provement of the growth by dipping the end of the Different types of molecular markers are used to explants in 0.1 mM glutathione before their setting in study genetic diversity in the genus of Pistacia – nutrient media. isozymes, RELP, RPD, etc. (Mehlenbacher, 2003, Sheibani and Villiers (1995) ascertain that the op- Vendramin at al., 2007). Their application for exam- timum media for propagation of P. vera, P. ining the philogenetic and taxonomic connections terebinthus and P. mutica is MS (Murashige and among the species is very important. Skoog, 1962) with 5 mg/1 BAP, and for rooting – In respect to the selection, markers for defining MS with 5 mg/l IBA. the sex of the young plants long before they achieve Other authors (Ghorbani et al., 2002) recommend reproductive age are in elaboration (Hormaza et al., for in vitro rooting of P. vera DKW media with ad- 1994; Kafkas et al., 2001). dition of 2 mg/l IBA and 0.01 mg/l NAA. In conclusion the study of the area of the rare trans- 452 P. Gercheva, A. Zhivondov, L. Nacheva and D. Avanzato

sexual form of Pistacia terebinthus and the common Chatibi, A., M. Kchouk, S. Thaminy, A. Ghorbel application of in vivo and in vitro methods for propa- and T. Khsib, 1998a. Multivariate analysis of the gation and molecular markers will allow its preserva- effects of growth regulators on the regeneration of tion and use as a rootstock and donor for mono- Pistacio (Pistacia vera L.) cv. Mateur from coty- eciousness in the selection programs of the pistachio. ledons. Acta Horticulturae, 470: 434-442. The potential for transfer of the monoeciousness Chatibi, A., M. Kchouk, S. Thaminy, A. Ghorbel feature to cultivars of P. vera can allow growing of and T. Khsib, 1998b. Multivariate analysis of the pistachio plantations without the necessary planting role of inorganic elements on micropropagation of of non-productive male trees for pollination and thus Pistacia (Pistacia vera L.) cv. Mateur. Acta at least 10 % increase of the crop can be achieved. Horticulturae, 470: 447-453. Crane, J. C., 1974.Hermaphroditsm in Pistacia. Cali- Acknowledgements fornia Agriculture, 28 (2): 3-4. This research is part of the project CC 16-07/2006, Gannoun, S., S. Lionakis and D. Gerasopoulos, supported by National Science Fund, Ministry of 1995 Aspects of in vitro culture of Pistacia Education and Science. terebinthus and Pistacia vera. Acta Horticulturae, 419: 201-206. Ghoraishi, S. R., 2006. Micropropagation of P. mutica. References Acta Horticulturae, 726: 183-192. Ghorbani, M., A. Azghandi, A. Sheibani and A. Abousalim, A. and S. Mantell, 1995. The ultrastruc- Esmailpour, 2002. Effects of altering culture me- ture of shoot apex of Pistacio (P. vera L.) grown in dium on rooting of Pistacio microshoots (Pistacia vitro with reference to shoot-tip necrosis. Acta vera cv.”Badami-Zarand”). Acta Horticulturae, Horticulturae, 419: 233-236. 591: 327-331. Avanzato, D., 2003 Una forma monoica di Pistacia Gonzales, A. and D. Frutos, 1990. In vitro culture terebinthus L scoperta in Bulgaria. Frutticoltura, of Pistacia vera L. embryos and aged tree explants. 10: 51-53. NATO ASI series A, 186: 335-338. Avanzato, D. and R. Quatra, 2004. Monoecious Hormaza, J., L. Dollo and V. Polito, 1994. Identifi- Pistacia terebinthus found in Bulgaria. Crop wild cation of a RAPD marker linked to sex determina- relative, 2: 14-16. tion in P. vera using bulked segregant analysis. Barghchi, M., P. Alderson and Y. Bajaja, 1989 Bio- Theor Appl Genet., 89: 9-13. technology in agriculture and forestry, vol.5 Trees Kafkas, S., 2002. Developing of monoecious Pistacio II, pp. 68-98. (P. vera L) populations and the sex determination Behboodi, B., 2002 Tissue culture results of all wild mechanism in Pistacia by cross-breeding. Acta Pistacio species and some cultivars in Iran Acta Horticulturae 591: 285-289. Horticulturae 591:399-403. Kafkas, S., S. Cetiner and R. Perl-Treves, 2001. Buffa, R., D Avanzato, A. Zhivondov, T. Caruso, Development of sex-associated RAPD markers in A. Vaccaro, F. P. Marra, M. Meli, T. Koceva, wild Pistacia species. J. Hort. Sci. Biotech., 76: T. Hristeva, E. Rea, A. Salerno and S. Rinaldi, 242-246. 2007. Phenological, morphological and physiologi- Kafkas, S., R. Perl-Treves and N. Kaska, 2000. cal aspects of Pistacia terebinthus L. genotypes Unusual Pistacia atlantica Desf. Monoecious sex native of Bulgaria with different asset as tree sexu- types in the Yunt Mountains of the Manisa prov- ality. In: A. Zhivondov (convener), First Balkan ince of Turkey. Isr. J. Plant Sci., 48 (4): 277-280. Symposium on Fruit Growing, 15-17 November Kaska, N., 2002. Pistashio nut growing in the 2007, Plovdiv, Bulgaria, p. 23. mediterranean basin. Acta Hort., 591: 443-451. Transsexual Forms of Pistachio (Pistacia terebinthus l.) from Bulgaria..... 453

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